Persistent activation of G?i/o-coupled receptors leads to enhanced adenylyl cyclase (AC) signaling that has been described using many different names, including cAMP overshoot and heterologous sensitization of AC. This adaptive response has been implicated in several psychiatric and neurological conditions. Previous studies support a hypothesis that persistent activation of G?i/o-coupled receptors promotes the dissociation/rearrangement of G? and G?? subunits in a pertussis toxin-sensitive manner that induces sensitization through a yet unknown mechanism. We hypothesize that drug-induced protein interactions with AC are responsible for the enhanced AC response. Previous studies have examined closely-related proteins or established AC interacting partners preventing the discovery of truly novel mechanisms. Thus, an unprecedented approach will be used to identify the sensitization interactome of adenylyl cyclase type 5 (AC5) in a neuronal cell model. These studies will use Bimolecular Fluorescence Complementation (BiFC) to perform cDNA library screening to identify sensitization-induced interacting proteins of AC5 in living cells. Specific am 1 will construct and characterize a neuronal cellular model for drug-induced BiFC of AC5. These studies will use CAD cells stably expressing an engineered AC5 fusion construct capable of fluorescence complementation with appropriate binding partners. Specific aim 2 shall construct the retrovirus-based BiFC cDNA library of potential AC5 binding partners for FACS screening. Specific aim 3 shall execute both primary and secondary screening for drug-induced BiFC. These studies will infect the neuronal cell model with the retroviral cDNA library followed by treatment with a G?i/o receptor agonist to induce heterologous sensitization of AC activity. Cells revealing drug-induced BiFC will be identified using FACS and isolated for cDNA amplification. Specific aim 4 will initiate a series of biochemical and functional studies to characterize those interacting proteins that represent the AC5 sensitization interactome. The anticipated outcome of these aims is the identification and characterization of novel AC5 interacting proteins relevant to heterologous sensitization. The impact of these scientific outcomes is substantial and will address a long-standing scientific question, develop a novel methodological approach, and have the potential to provide drug targets for in vivo studies.